HIV diagnostics are essential for both diagnosis and management of HIV infection. There have been many advances in the testing for HIV over the past decade and we are now able to detect HIV much faster in the latest generation of testing methodologies.


To answer this question, we have to first reconcile with the concepts of eclipse period and window period and also the various antigens and antibodies that are the target of HIV assays.

Eclipse period

After an exposure that leads to infection, there is a variable amount of time called the eclipse period in which no existing diagnostic test is capable of detecting HIV – this is known as the eclipse period (Figure1).(1)

Figure 1 – Timeline of virological and serological events following an HIV infection(1)

Window period

The time period between infection and the first reactive result on a given test is called the window period, the length of which depends on the target being detected by a particular assay. For example, tests capable of identifying anti-HIV IgM have a shorter window period compared with those detecting only IgG.(1)


HIV tests have been typically classified into generations – each improving incrementally on its predecessor(s) in terms of performance and shortening of the window period.(1)

Table 1 – Generation of anti-HIV antibody tests(2–4)

Generation Window period Detects Antibody or Antigen (Ag)
1st generation ~ 8-10 weeks IgG Anti HIV-1
2nd generation ~ 4-6 weeks IgG Anti HIV-1 & IgG Anti HIV-2
3rd generation ~ 2-3 weeks IgG and IgM anti HIV-1, HIV-2 and HIV Group O
4th generation ~ 2 weeks IgG and IgM anti HIV-1, HIV-2 and HIV Group O.

Also detects HIV-1 p24 Ag


The median window period for 4th generation tests is 17.8 days, whereas it is 23.1 days for the 3rd generation assays.(5)

There are some tests which are informally called 5th generation tests, but are not officially recognized as such because there is no reduction of window period from the 4th generation. These tests can also distinguish between samples positive for p24 antigen and for antibodies, and also between HIV-1 and HIV-2.(6)

Diagnosing HIV in asymptomatic patients(2)




Assays A1, A2, A3 represent 3 different assays based on different principles or different antigenic compositions. Assay A1 should be of high sensitivity and A2 and A3 should be of high specificity. A2 & A3 should also, ideally, be able to differentiate between HIV 1 & 2 infection.(2)

In case of an indeterminate result, testing should be repeated on a second specimen taken after 14-28 days. In case the serological results continue to be indeterminate, then the specimen is to be subjected to PCR if facilities are available or refer to the National Reference Laboratory for further testing. (2) If the HIV-1 PCR is positive, it is confirmed as positive. If negative, do HIV-2 PCR testing to confirm or rule out HIV-2.

Diagnosing HIV in a patient with AIDS-indicator-disease symptoms(2)

Diagnosing HIV in a patient – Sentinel surveillance(2)

This type of HIV surveillance testing is anonymous and unlinked – hence post-test counselling is not possible. (2)

Screening for blood transfusion and transplant(2)

The specimen is subjected to one test for HIV reactivity. The test used must have high sensitivity. If non reactive, the specimen is considered free of HIV (negative) and if reactive, the specimen is considered as HIV positive. This strategy is used for ensuring donation safety (e.g., blood, blood products, organs, tissues, sperms etc.). The unit of blood that tests reactive (positive) is to be discarded.(2)

If the donor is to be notified of his result, based on his prior consent, it becomes a matter of diagnosis (in which case one of the above algorithms must be used after proper counselling and the donor should be referred to an ICTC (Integrated Counselling and Testing Centre).(2)

Diagnosis of HIV in infants with HIV positive mothers

HIV seropositivity in a child younger than 18 months is not diagnostic (because a positive antibody test result during the first 18 months of life could represent either persistent maternal antibody in an HIV–uninfected child or antibody newly produced by an HIV–infected child. Ergo, serologic testing of a child younger than 18 months cannot be used to diagnose HIV infection and virologic assays are required. However, a positive antibody test result in the infant generally indicates maternal HIV infection.(7)

It has been recommended that diagnostic testing with HIV RNA assays be performed: (7)

  • within the first 14 days of life, AND.(1)
  • at 1 to 2 months of age, AND(1)
  • at 3 to 6 months of age.(1)

If any of these test results are positive, repeat testing is recommended to confirm the diagnosis of HIV infection.(7) A diagnosis of HIV infection can be made on the basis of 2 separate positive HIV RNA assay results. (7)


The latest Ag/Ab combination assays have been very successful screening tools – having reduced the window period and succeeded in identifying 82% of the antibody-negative infections which would otherwise only be detectable by HIV RNA PCR. False-positives have come down to less than 2 per 100,000. But there is always room for improvement.(5)

One suggestion by Branson et al (2019) is to perform a quantitative HIV-1 viral load for every patient with a reactive 4th generation test. This would contribute to confirm the diagnosis and in the future clinical management.(5) In his post in the New England Journal of Medicine Journal Watch blog, Dr. Paul Sax (clinical director of the infectious diseases division at Brigham and Women’s Hospital, Boston, MA) concurs with this view.(8)

A viral load, no matter how low is confirmatory of the diagnosis and treatment can be initiated immediately, which helps in reducing HIV transmission, preserving immune function, and possibly providing the best candidates for HIV cure strategies when they become available in the future.(8)

A negative viral load would indicate the patient is one of the following:
It has been recommended that diagnostic testing with HIV RNA assays be performed: (7)

  • Patient is an “elite controller”. Elite controllers are a small subset of people living with HIV who have sustained antibody levels but are able to maintain suppressed viral loads for years without antiretroviral therapy (ART). (8,9)
  • “Patient on antiretroviral therapy who has not disclosed their diagnosis(1)
  • “Patient is of those rare individuals who is HIV-2 positive.(8)
  • “Patient had a false positive HIV screen(8)

All of these can be further differentiated on doing HIV1/2 antibody differentiation tests.

Figure 3 – Suggested Reflex Viral Load Testing Algorithm(5,8,10)


Suburban Diagnostics performs HIV testing using the following methodologies

  • Chemiluminescent microparticle immunoassay (CMIA) on the Abbott – Architect i1000SR: This is a 4th generation test detecting HIV p24 antigen and antibodies to HIV type 1 (HIV-1 group M and group O) and/or type 2 (HIV-2).(1)
  • Electrochemiluminescence Immunoassay (ECLIA) on the Roche Cobas e411: This is a 4th generation test detecting HIV p24 antigen and antibodies to HIV type 1 (HIV-1 group M and group O) and/or type 2 (HIV-2).(1)
  • Immunofiltration on the HIV-Tridot: This is a 3rd generation rapid screening test detecting antibodies to HIV-1/2..(1)
  • Immunochromatography on the Alere Determine Card Test: This is a 3rd generation rapid screening test detecting antibodies to HIV-1/2.Immunofiltration on the HIV-Tridot: This is a 3rd generation rapid screening test detecting antibodies to HIV-1/2.(1)


On the molecular front, for HIV-RNA, Suburban Diagnostics uses the fully automated FDA approved platforms from Roche – The Cobas Ampliprep / Cobas Taqman HIV-1 test, Version 2.0 for HIV-1 RNA monitoring of patients infected with group M & group O HIV -1 virus. The two targets (HIV-1 gag & LTR) specific dual labelled probes ensures a complete coverage of Group M subtypes as well as Group O of HIV-1 RNA.


  1. Hurt CB, Nelson JAE, Hightow-Weidman LB, Miller WC. Selecting an HIV Test: A Narrative Review for Clinicians and Researchers. Sex Transm Dis. 2017 Dec;44(12):739–46.
  2. National Guidelines for HIV Testing [Internet]. National AIDS Control Organization, Ministry of Health & Family Welfare, Government of India; 2015 [cited 2019 Nov 26]. Available from:
  3. Alexander TS. Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution. Clin Vaccine Immunol. 2016 Apr 1;23(4):249–53.
  4. Blog LBP. Best Practices for HIV-1/2 Screening: When to Test and What to Test [Internet]. Laboratory Best Practice Blog. [cited 2019 Nov 28]. Available from:
  5. Branson BM. HIV Diagnostics: Current Recommendations and Opportunities for Improvement. Infect Dis Clin. 2019 Sep 1;33(3):611–28.
  6. A “fifth generation” HIV assay? [Internet]. Medical Laboratory Observer. [cited 2019 Nov 26]. Available from:
  7. Read JS, and the Committee on Pediatric AIDS. Diagnosis of HIV-1 Infection in Children Younger Than 18 Months in the United States. PEDIATRICS. 2007 Dec 1;120(6):e1547–62.
  8. Our HIV Testing Algorithm Has a Major Problem – Here’s How to Fix It [Internet]. HIV and ID Observations. 2019 [cited 2019 Nov 27]. Available from:
  9. Elite Controllers Definition [Internet]. AIDSinfo. [cited 2019 Nov 27]. Available from:
  10. Considerations for the clinical management of HIV elite controllers | The Ontario HIV Treatment Network [Internet]. [cited 2019 Nov 27]. Available from: